骨髓间充质干细胞为核供体的克隆绵羊的生产与鉴定

本研究旨在采用骨髓间充质干细胞（BMSCs）为核供体进行体细胞核移植,以获得成体干细胞克隆绵羊。选择幼体绵羊BMSCs为核供体,构建重构胚进行克隆胚胎移植,克隆绵羊诞生后,选取IASG上公布的绵羊10个微卫星标记位点,PCR扩增克隆羊、供体细胞及代孕母羊微卫星DNA,利用Quantity One软件进行基因分型,计算供体细胞与克隆羊之间的父子关系相对机会（RCP）值。结果显示,以幼体绵羊BMSCs为核供体,与去核成熟卵母细胞进行核移植,构建重构胚进行电融合,其融合率达80.62%,重构胚发育至桑椹胚阶段,经手术植入20只代孕母羊,获得克隆绵羊5只,成活3只。克隆羊基因型与供体细胞一致,供体细胞与克隆羊之间的RCP≥99.999%,证明克隆羊来自幼体绵羊BMSCs。本试验最终成功获得了首例以BMSCs为核供体的克隆绵羊。     

微卫星与金华猪生长性状关系的研究

采用微卫星标记多重扩增结合荧光标记检测技术,对152头金华猪种群的30个微卫星位点的不同基因型与生长发育性状的关系进行了初步分析和显著性检测。结果显示SW 2476、SW 0215、SW 24等3个微卫星位点不同基因型间的出生重差异显著;SW 902、S0143、SW 0215、S0228、SW 24等5个微卫星位点不同基因型间的60日龄重差异显著;S0178、S0218、S0255、S0155、SW 951等5个微卫星位点不同基因型的4月龄重差异显著;而S0217微卫星位点的不同基因型的6月龄重差异显著。     

克隆清原马鹿的微卫星鉴定

为对体细胞克隆马鹿进行遗传鉴定,选取8对在牛中具有多态性的微卫星位点,通过筛选出在清原马鹿中具有高度多态性的引物,分别对体细胞克隆清原马鹿、清原马鹿供体细胞、受体清原马鹿进行微卫星分析。结果表明:清原马鹿的HUJ1177、BM888、BM757、IDVGA37、oarFCB304、RM12等6个微卫星位点的PCR扩增产物经聚丙烯酰胺凝胶电泳后呈现多态性。经PAGE电泳分析,克隆清原马鹿与供体细胞的微卫星DNA基因型完全相同,而与受体清原马鹿的微卫星基因型明显不同。因此,体细胞克隆清原马鹿基因组来源于供体清原马鹿细胞而与受体无亲缘关系;HUJ1177、BM888、BM757、IDVGA37、oarFCB304、RM12等6个微卫星位点可用于克隆清原马鹿的鉴定。     

辽宁绒山羊微卫星多态性及其与经济性状相关性研究

根据山羊遗传连锁图谱和相关资料,选用7个微卫星座位,对40只辽宁绒山羊基因组DNA的遗传多样性进行检测;通过标记多态性与生产性状的最小二乘线性模型,进行分子标记与经济性状的相关分析.结果表明,7个微卫星座位中只有4个表现出了多态性.微卫星OarJMP8座位上AA、AD基因型个体羊绒纤维长度显著高于其它基因型个体(P<0.05),AD基因型个体产绒量显著高于其它基因型个体(P<0.05);微卫星BM143座位上AC基因型个体羊绒纤维细度显著低于其它基因型个体(P<0.05);微卫星OarAE101座位上AB基因型个体羊绒纤维长度和产绒量均显著或极显著高于其它基因型个体(P<0.05,P<0.01);微卫星BM6506座位上从基因型个体产绒量极显著高于其它基因型个体(P<0.01).     

丝羽乌骨鸡月产蛋性能与微卫星标记关系的研究

本研究首次从月产性能这个角度对丝羽乌骨鸡的产蛋性能进行微卫星标记分析;从鸡的遗传图谱中选择10个与产蛋性能相关的微卫星基因座,随机选取丝羽乌骨鸡共120只进行标记基因型与产蛋性状的最小二乘分析,结果表明有5个标记分别与4个不同的月产蛋性状间存在显著相关.其中标记MCW0170的基因型DE和标记MCW0241的基因型AA最有望作为月产蛋量及早期产蛋性能的辅助标记.     

陶赛特羊Carwell基因微卫星标记多态性与体重和体尺指标的相关性分析

据相关研究报道,在澳洲无角陶赛特羊18号染色体末端微卫星标记CSSM18和TGLA122之间的区域有控制其肌肉生长发育的Callipyge和Canvell基因。研究选择了微卫星标记CSSMl8和TGLAl22之间的TGLA122、LS33、ILSTS54、MCMA26四个微卫星标记,测定了新疆引进的澳洲无角陶赛特羊所产后代163只羔羊早期生长发育性状指标,采用PCR—SSCP技术检测了上述4个标记在研究群体中的多态性,分析了研究群体中4个微卫星标记不同基因型与早期生长发育性状的相关性。结果显示：微卫星标记TGLA122的杂合度0．4301,多态信息含量0．2733,有效等位基因数1．3761;微卫星标记MCMA26的杂合度0．4824,多态信息含量0．3660,有效等位基因数1．577O;微卫星标记LS33的杂合度0．3283,多态信息含量0．2744,有效等位基因数1．3780。微卫星标记TGLA122的3种基因型（AA／BB／AB）对90日龄母羔臀宽影响显著（P〈0．05）,对其他生长指标均无显著影响;微卫星标记MCMA26的3种基因型（GG／Tr／GT）对60日龄母羔胸围和臀宽有显著影响,对其他生长指标均无显著影响;微卫星标记LS33的2种基因型（EE／EF）个体的体重和体尺均无显著性差异;微卫星标记ILSTS54在该群体中无多态性。     

辽宁绒山羊微卫星多态性及其与经济性状相关性研究

研究分析了辽宁绒山羊3个经济性状的相关性.根据山羊遗传连锁图谱和相关资料,选用7个微卫星座位,对40只辽宁绒山羊基因组DNA的遗传多样性进行检测,通过标记多态性与生产性状的最小二乘线性模型,进行分子标记与经济性状的相关分析.结果表明:7个微卫星座位中只有4个表现出了多态性.微卫星OarJMP8座位上AA、AD基因型个体羊绒纤维长度极显著高于其他基因型个体(P＜0.01),AD基因型个体产绒量显著高于其他基因型个体(P＜0.05);微卫星BM143座位上AC基因型个体羊绒纤维细度显著低于其他基因型个体(P＜0.05);微卫星OarAE101座位上AB基因型个体羊绒纤维长度和产绒量均显著高于其他基因型个体(P＜0.05);微卫星BM6506座位上从基因型个体产绒量极显著高于其它基因型个体(P＜0.01).     

安徽白山羊保种群微卫星标记遗传多样性分析

为了解安徽白山羊保种群的遗传多样性,试验选取了具有较高多态性的7个微卫星标记,进行了安徽白山羊保种群的遗传多样性分析。结果表明:7个微卫星标记共检测到45个等位基因和54种基因型,以OarAE54等位基因和基因型数量最多,DRBP1等位基因和基因型数量最少,每个标记的平均等位基因数为6.43个;7个微卫星标记的多态信息含量和平均杂合度均高于0.85,显示为高度多态;OarAE54标记的群体遗传杂合度高达0.864。说明安徽白山羊保种群群体遗传多样性丰富。     

供体细胞对绵羊转基因体细胞克隆胚胎体外发育影响的研究

研究比较了转入不同外源基因、转同一基因的不同细胞克隆、供体细胞的培养代数及冷冻对转基因体细胞克隆羊重构胚体外发育的影响,旨在为进一步探寻建立供体细胞个性化准备方案,为提高动物克隆效率提供参考。结果表明,转不同外源基因的供体细胞对转基因体细胞克隆羊重构胚体外发育无影响;转同一基因的不同细胞克隆对转基因体细胞克隆羊重构胚体外发育有一定影响,且转TERT基因的不同克隆间的重构胚的融合率及囊胚率差异显著(P0.05)。随着供体细胞培养代数的增加,重构胚体外发育的桑葚胚率及囊胚率无显著影响,但融合率随着培养代数的增加有上升趋势,且差异显著(P0.05);供体细胞冷冻可促进其融合率、桑葚胚率及囊胚率。研究结果表明,供体细胞的不同克隆、培养代数、冷冻等因素对转基因效率有一定的影响。     

荷斯坦牛亲子鉴定检测方法的建立与应用

为了构建北京奶牛中心荷斯坦种公牛个体及亲子鉴定DNA数据库,本研究从联合国粮农组织(FAO)和国际动物遗传学会(ISAG)推荐的具有高度多态性的微卫星标记中选取11个常染色体微卫星标记(BM1824、BM2113、ETH3、ETH10、ETH225、INRA023、SPS115、TGLA53、TGLA122、TGLA126和TGLA227),采用荧光引物PCR和ABI3730XL遗传分析仪,对229头荷斯坦种公牛个体基因型进行了检测,并探讨其用于牛个体识别和亲子鉴定的可行性。研究结果表明,11个微卫星标记平均杂合度为0.733,平均多态信息含量为0.695,其中TGLA122标记平均多态信息含量最高为0.866,SPS115标记最低为0.506,均属于高多态性标记。11个标记累积个体识别能力为99.99%,累积非父排除率达到99.99%,随机个体一致性概率为1.59×10-17。研究结果表明上述11个微卫星标记进行个体识别和亲子鉴定的效力与可靠性均很高,同时初步建立了北京奶牛中心荷斯坦种公牛DNA数据库。     

Mice cloned by nuclear transfer from somatic and ntES cells derived from the same individuals

The current success rate of cloned mice from adult somatic cell nuclei is very low, whereas it is relatively high for cloned mice from ES cell nuclei. In this experiment, we examined whether the success rate of cloning from somatic cells could be improved via nuclear transfer embryonic stem cells (ntES cells) established from somatic cell nuclei. We obtained 11 cloned mice and 68 ntES cell lines from the somatic cell nuclei of 7 mice, and cloned 41 mice were cloned from the ntES cell nuclei. Unexpectedly, the overall success rate of cloning from ntES cell nuclei in this series was no better than when using somatic cell nuclei. Interestingly, full-term cloned mice were produced only via ntES cells from two individuals, but not by direct nuclear transfer from the somatic cells, and vice versa. Ultimately, we were able to obtain clone mice from 6 out of 7 individuals using either somatic cells or ntES cells. Thus, although ntES cells as donor nuclei do not absolutely assure a better success rate for mouse cloning than somatic cells, to preserve and clone valuable individuals, we recommend that ntES cell lines be established. These can then be used as an unlimited source of donor nuclei for nuclear transfer, and thus complement conventional somatic cell nuclear transfer cloning approaches.     

Production and Identification of Clone Sheep Using Bone Marrow Mesenchymal Stem Cells as Donor Cell

In order to obtain the cloned sheep using bone marrow mesenchymal stem cells（BMSCs）as the donor cells,the BMSCs of sheep were chosen and reconstructed embryos were built to transfer.10 published microsatellite markers were chosen,and the DNA samples from clone sheep,donor cells and surrogate ewes were amplified,and the relationship of father-son（RCP）was analyzed using the Quantity One for genotyping.The results showed that the reconstructed embryos were successfully built for electric fusion using sheep BMSCs as nuclear donor,and making nuclear transplantation into enucleated mature oocytes of which the fusion rate was 80.62%.20 surrogate ewe were chosen to be implanted with the reconstructed embryos at morula stage by implant surgery,and 5 lambs were born and only 3 were survived.The genotype of cloned sheep was in line with the dornor cell and the RCP were more than 99.999%.In conclusion,the first clone sheep were obtained successfully by using BMSCs as a nuclear donor in this experiment.     

Propagation of elite rescue dogs by somatic cell nuclear transfer

The objective of the present study was to compare the efficiency of two oocyte activation culture media to produce cloned dogs from an elite rescue dog and to analyze their behavioral tendencies. In somatic cell nuclear transfer procedure, fused couplets were activated by calcium ionophore treatment for 4 min, cultured in two media: modified synthetic oviduct fluid (mSOF) with 1.9 mmol/L 6‐dimethylaminopyridine (DMAP) (SOF‐DMAP) or porcine zygote medium (PZM‐5) with 1.9 mmol/L DMAP (PZM‐DMAP) for 4 h, and then were transferred into recipients. After embryo transfer, pregnancy was detected in one out of three surrogate mothers that received cloned embryos from the PZM‐DMAP group (33.3%), and one pregnancy (25%) was detected in four surrogate mothers receiving cloned embryos from the SOF‐DMAP group. Each pregnant dog gave birth to one healthy cloned puppy by cesarean section. We conducted the puppy aptitude test with two cloned puppies; the two cloned puppies were classified as the same type, accepting humans and leaders easily. The present study indicated that the type of medium used in 6‐DMAP culture did not increase in cloning efficiency and dogs cloned using donor cells derived from one elite dog have similar behavioral tendencies.     

体细胞克隆技术在乌金猪遗传资源保护上的应用

旨在通过猪体细胞克隆技术,生产乌金猪火毛系,并分析生长发育能力及繁殖性能,为该技术在地方优良猪种保种上的应用提供理论依据。本研究采集了符合乌金猪种质特征的3头公猪和12头母猪的耳组织样品。公猪年龄分别为3、6、11月龄,母猪年龄差距较大,最小的2月龄,最大的10岁。其中3头母猪和1头3月龄公猪已被去势,10岁母猪已无繁殖能力。结果,成功建立了15头乌金猪的耳组织成纤维细胞系,分别选择1头3月龄乌金猪（♂）和10岁乌金猪（♀）的成纤维细胞进行体细胞核移植,经胚胎移植入16头代孕母猪,共获得25头克隆猪,克隆猪生长发育正常,性成熟后进行自然交配共获得39头F1代活仔。本研究通过体细胞克隆技术成功克隆了乌金猪,具备正常的生长发育性能和繁殖性能,为地方猪种的保护研究提供了新的途径。     

Application of Somatic Cell Cloning Technology in the Protection of Genetic Resources of Wujin Pigs

The purpose of this experiment was to study the growth and developmental ability and reproductive performance of cloned Wujin pigs fire hair line by somatic cell cloning technology, and provide a theoretical basis for the application of somatic cell cloning technology in the conservation of local excellent pig breeds. In this study, the ear samples were collected from 3 male and 12 female pigs which met the characteristics of Wujin pigs. The 3 boars were 3, 6 and 11 months old, respectively. The sows had a large age gap, from 2 months old to 10 years old, the three sows and one boar of 3-month-old had been castrated and the sow of 10-year-old had no reproductive capacity. The ear fibroblast cell lines were successfully established from 15 Wujin pigs. Then the fibroblast cells from 3-month-old(♂) and 10-year-old(♀) pigs were used as donor cells for somatic cell nuclear transfer. The cloned embryos were transplanted into 16 surrogate sows and 25 cloned pigs were obtained. The growth and development indexes of these cloned pigs were all normal. After sexual maturity, the male and female cloned pigs mated naturally and 39 live piglets (F1) were obtained. This study successfully cloned Wujin pigs by somatic cell nuclear transfer technology, and cloned Wujin pigs had normal growth and developmental ability and reproductive performance. The study result provides a new way for the conservation of local pig breeds.     

动物体细胞克隆技术的研究进展

体细胞克隆绵羊“多莉”(Dolly)的诞生,表明成年哺乳动物体细胞具有基因组的全能性。本文从“多莉”诞生的背景、体细胞克隆技术的研究现状、体细胞核移植过程中的核质互作、该技术目前存在的问题及其在制作转基因动物上的应用等方面,概述了体细胞克隆技术的研究进展。     

Effects of donor cells’ sex on nuclear transfer efficiency and telomere lengths of cloned goats

The aim of this study was to investigate the effects of donor cells’ sex on nuclear transfer efficiency and telomere length of cloned goats from adult skin fibroblast cells. The telomere length of somatic cell cloned goats and their offspring was determined by measuring their mean terminal restriction fragment (TRF) length. The result showed that (i) reconstructed embryos with fibroblast cells from males Boer goats obtained significantly higher kids rate and rate of live kids than those of female embryos and (ii) the telomere lengths of four female cloned goats were shorter compared to their donor cells, but five male cloned goats had the same telomere length with their donor cells, mainly due to great variation existed among them. The offspring from female cloned goats had the same telomere length with their age‐matched counterparts. In conclusion, the donor cells’ sex had significant effects on nuclear transfer efficiency and telomere lengths of cloned goats.     

供体细胞对猪体细胞克隆胚胎早期发育的影响

以中国农业大学实验用小型猪香猪胎儿成纤维细胞、成年耳成纤维细胞和颗粒细胞3种细胞系为供体细胞进行核移植。比较了血清饥饿法和接触抑制法处理胎儿成纤维细胞诱导进入G0／G1期的效率，发现二者差异不显著（P〉0．05），血清饥饿2d和4d差异不明显，同样接触抑制2d和4d差异也不显著（P〉0．05）。系统研究了影响克隆胚胎发育的供体因素：血清饥饿与否、细胞形态、细胞类型及个体差异等，结果表明：血清饥饿处理对克隆胚的早期发育没有明显的促进作用；圆形光滑细胞有利于细胞融合，对早期发育无显著影响（P〉0．05）；不同个体、不同类型的供体细胞对克隆胚囊胚发育率有一定的影响。     

Vitamin C treatment of embryos,but not donor cells,improves the cloned embryonic development in sheep

Vitamin C is not only an antioxidant but also a regulator of epigenetic modifications that can enhance the activity of the ten-eleven translocation (TET) family dioxygenases and promote the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Here, we investigated the effects of vitamin C in regulating DNA methylation in sheep somatic cells or embryos in an effort to improve the cloned embryo development. Vitamin C treatment of sheep foetal fibroblast cells significantly increased the 5hmC levels but did not affect the 5mC levels in cells. After nuclear transfer, vitamin C-treated donor cells could not support a higher blastocyst development rate than non-treated cells. Although combination of serum starvation and vitamin C treatment could induce significant 5mC decrease in donor cells, it failed to promote the development of resultant cloned embryos. When cloned embryos were directly treated with vitamin C, the pre-implantation development of embryos and the 5hmC levels in blastocysts were significantly improved. This beneficial role of vitamin C on embryo development was also observed in fertilized embryos. Our results suggest that vitamin C treatment of the embryos, but not the donor cells, can improve the development of cloned sheep embryos.